The importance of protein phosphorylation as a fundamental mechanism that controls cell physiology was established by the pioneering work of Krebs and Fisher (Fed. Proc., 1966, 25, 1511-1520). Protein kinases modify protein function by transferring phosphate groups from ATP or GTP to free hydroxyl groups of amino acids. Most protein kinases phosphorylate serine and threonine residues, but a subset of protein kinases selectively phosphorylate tyrosine residues. There are 90 protein tyrosine kinases (PTKs) which play specific roles (Hunter, Cell, 1987, 50, 823-829). PTKs can be further divided into the two main subgroups, receptor tyrosine kinase (RTKs) and non-receptor, cytosolic tyrosine kinases. RTKs contain an extracellular ligand binding domain, transmembrane region and intracellular cytoplasmic kinase domain. PTKs have a conserved kinase domain structure that consists of an N-terminal lobe (N-lobe) composed of a five-stranded β-sheet and a single α-helix, connected to a larger C-terminal lobe (C-lobe) by a hinge region. The protein substrate binds to a surface groove formed by the α-helical C-lobe, and an ATP binding pocket is formed by the hinge region and N- and C-lobes. The C-lobe contains the activation loop (A-loop), which becomes phosphorylated. Phosphorylation leads to the conformational stabilization and activation, allowing the transfer of the γ-phosphate from bound ATP to the bound substrate protein. All protein kinases share structural similarities but show greater structural distinction in their inactive state (Schinlder et al., Science, 2000, 289, 1938-1942).
Janus Kinases (JAKs) are non-receptor tyrosine kinases and were discovered in searches for novel protein tyrosine using PCR based strategies (Firmbach-Kraft et al., Oncogene, 1990, 5, 1329-1336 and Wils A F, Proc. Natl. Acad. Sci. USA, 1989, 86, 1603-1607). In mammalians, JAKs have four members JAK1, JAK2, JAK3 and TYK2 enzymes. Since the sequencing of other vertebrate genomes has been completed, we know that there are four JAK family members in mammals, birds and fishes. In humans, the JAK1 gene is located on chromosome1p31.3 and JAK2 is on 9p24; JAK3 and TYK2 genes are clustered together on chromosome19p13.1 and 19p13.2, respectively. The three-dimensional structure of the JAKs is at present unknown but seven JAK homology (JH) domains have been identified, numbered from carboxyl to the amino terminal. The JH1 domain at the carboxyl terminal has all the features of a typical eukaryotic tyrosine kinase domain. Interestingly, this domain is most closely related to the kinase domains of the epidermal growth factor family of receptor tyrosine kinases, suggesting that JAK family may have arisen from the larger family of protein kinases (Manning et al., Science, 2002, 298, 1912-1934).
In mammals, JAK1, JAK2 and TYK2 are ubiquitously expressed. In contrast the expression of JAK3 is more restricted; it is predominantly expressed in hematopoietic cells and is highly regulated the cell development and activation. At the cellular level, JAKs can be found in the cytosol when they are experimentally expressed in the absence of cytokine receptors, but, because of their intimate association with cytokine receptors, they ordinarily localize to endosome and the plasma membrane, along with their cognate receptors (Kawamura et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 6374-6378 and Musso et al., J. Exp. Med., 1995, 181, 1425-1431). A large number of cytokines are dependent upon JAK1, including a family that use a shared receptor subunit called common γ chain (γc), which includes interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15 and IL-21. These cytokines are also dependent on JAK3, because JAK3 binds γc. JAK1 is also essential for another family that uses the shared receptor subunit gp130 (IL-6, IL-11, oncostatin M, leukemia inhibitory factor (LIF), ciliary neutrophilic factor (CNF) as well as granulocyte colony-stimulating factor (G-CSF) and IFNs. JAK2 is essential for the hormone-like cytokines such as growth hormone (GH), prolactin (PRL), erythropoietin (EPO), thrombopoietin (TPO) and the family of cytokines that signal through the IL-3 receptor (IL-3, IL-5 and GM-CSF). JAK2 is also important for cytokines that use the gp130 receptor and for some IFNs.
JAK3 probably has the most discrete function, as is associated with only one cytokine receptor-the common gamma chain or γc. This is a shared receptor subunit that pairs with other ligand-specific subunits to form the receptors for IL-2, IL-4, IL-7, II-9, IL-15 and IL-21. The immune abnormalities associated with JAK3 deficiency were confirmed with the generation of JAK3 knockout mice; similar to humans, these mice have SCID that resembles γδ deficiency with small thymuses, absence of lymph nodes and reduced numbers of α/β and γ/δ T-cells. JAK3 mice also have a profound reduction in thymic progenitor cells and reduced ability to reconstitute T cell development. No effect on myeloid or erthyroid cells was noted in JAK3 deficient mice consistent with observations in humans and indicative of a specific effect on lymphoid precursors. However, murine JAK3 deficiency is associated with a T-cell dependent autoimmune disease characterized by infiltration of tissues by mononuclear cells, splenomegaly, and expansion of neutrophils and monocytic cells. These auto-immune diseases are T-cell dependent.
TYK2 was the first JAK to be implicated in IFN signaling, but subsequent studies indicate that TYK2 is essential for IL-12, IL-6, and IL-10 signaling, but not for cytokines that use gp130. IL-12 induced IFN-γ production by NK cells and activated T-cells was highly dependent on TYK2. Infact, TYK2 was shown to be involved in IL-23 induced STAT3 phosphorylation of activated T-cells and in IFN-γ production. Furthermore, TYK2 plays a crucial role in IL-23 induced IL-17a production by γδ T cells. The importance of TYK2 in the in vivo differentiation of Antigen-specific Th1 cells has also not been defined. TYK2 mice also have defective responses to lipopolysaccharide (LPS, a component of the outer membrane of gram-positive bacteria), but whether this is a direct or indirect effect has not been defined. In particular, a role of TYK2 in signaling through the Toll receptor, which mediates the response to LPS, has not been established (Shimoda et al., Immunity, 2000, 13, 561-571; Karaghiosoff et al., Immunity, 2000, 13, 549-560).
JAKs are continuously associated with the membrane-proximal regions of cytokine receptors, although in some cases interaction between the JAK and the receptor is increased upon ligand binding. It has been proposed that ligand binding brings a conformational change in the receptor, which promotes JAK activation through reciprocal interaction of two juxtapositioned JAK kinases and auto-and/or trans-phosphorylation of tyrosine residues on the activation loop of the JAK kinase domain. Like other tyrosine kinases, JAKs undergo autophosphorylation, but the importance of this modification in JAK-dependent signaling is not very well understood. Autophosphorylation within the activation loop positively regulates kinase activity; in JAK3, however, phosphorylation in this region can enhance or inhibit catalytic activity, depending upon the site of phosphorylation. Other sites of phosphorylation have recently been identified, e.g., a conserved residue in the hinge region between JH1 and JH2 is a prominent site of auto-phosphorylation (Tyr813 in JAK2 and Tyr785 in JAK3). This site serves to recruit the adapter protein Sh2-Bb, which positively regulates JAK2 activity (Feener et al., Mol. Cell Biol., 2004, 24, 4968-4978).
Tyrosine phosphorylation of cytokine receptors provides binding sites for signaling molecules. The main family of DNA-binding signaling proteins with transcriptional activity responsible for mediating signals from cytokine receptors is the STAT family. The seven members of the mammalian signal transducer and activator of transcription (STAT) family participates in a wide range of biological processes with impact both on the generation and the functional regulation of the cells involved in immunity. STATs bind phosphorylated receptors and in turn are substrates for the JAKs. Phosphorylated STAT can interact, dimerize, traffic into the nucleus, and regulate gene expression. The mechanisms by which cytokine activation of a restricted number of JAKs results in such different and specific downstream signaling events are still not understood. A large number of cytokines uses only four PTKs and seven STAT proteins for their specific signaling transmission. Activation of STATs by JAKs is a hallmark of both innate and adaptive immune responses. Hence, lymphocyte development, survival, and proliferation resulting in the outcome of an immune response by T-helper (Th) cell lineage-defining cytokines all depend on JAK activation as a primary step. Genetic analysis of JAKs in human with certain immune diseases and the generation of JAK-specific knockout (KO) mice helped in understanding the exact role of JAKs in immune cell signaling (Darnell et al., Science, 1994, 264, 1415-1421; Ihle et al., Annu. Rev. Immunol., 1995, 13, 369-398; Leonard et al., Ann. Rev. Immunol., 1998, 16, 293-322; Liu et al., Curr. Opin. Immunol., 1998, 10, 271-278 and Levy et al., Nat. Rev. Mol. Cell Biol., 2002, 3, 651-662).
Cytokines are crucial for development, survival, proliferation and differentiation of hematopoietic cells. Type I and type II cytokine receptors lack receptor-intrinsic tyrosine kinase activity and instead transmit their signals through receptor associated JAKs. In principle, all four JAKs might be considered as useful therapeutic targets.
Due to T-cell dependencies and their Th1 and Th17 modulation activities TYK2 enzyme inhibitors may have roles in auto-immune disorders such as rheumatoid arthritis, multiple sclerosis, psoriasis, intestinal bowel disease, and also in a few inflammatory disorders such as chronic obstructive pulmonary disease or allergy. A fully human monoclonal antibody targeting the shared p40 subunit of the IL-12 and IL-23 cytokines was recently approved by the European Commission for the treatment of moderate to severe plaque psoriasis (Krueger et al., N. Engl. J. Med., 2007, 356, 580-92, and Reich et al., Nat. Rev. Drug Discov., 2009, 8, 355-356). In addition, an antibody targeting the IL-12 and IL-23 pathways underwent clinical trials for treating Crohn's disease (Mannon et al., N. Engl. J. Med., 2004, 351, 2069-79).
Many pharmaceutical companies have established JAK targeted drug development programs, including Pfizer, Vertex, Rigel and Incyte. Pfizer has progressed CP-690550, which is a potent JAK3 inhibitor. This compound was reported to show efficacy in an animal model of organ transplantation and clinical trials, however the molecule is not selective for JAK3 and also inhibits JAK2 kinase with almost equipotency.
WO2010/142752 and US2010/0317643 disclose compounds having TYK2 inhibitory activity. DE102009001438 and DE102009015070 disclose carbonylamino substituted anilino pyrimidine derivatives as TYK2 inhibiters. WO2011/113802 discloses imidazopyridine compounds useful for treating diseases mediated by TYK2 kinase. US2010/0311743 discloses the compounds which are inhibitors of protein kinases, particularly JAK family kinases. US2010/0256365 discloses compounds which are active on protein kinases. WO2005/082367 discloses compounds having kinase inhibitory activity.
WO2005/097129 discloses 6-azaindole compounds having superior IκB kinase inhibitory activity. US2005/0228000 discloses bicyclic heterocycles useful as serine protease inhibitors. US2010/063047 discloses inhibitors of histamine receptor. WO2009/129335 discloses Inhibitors of histone deacetylase. WO2005/028434 discloses compounds which are inhibitors of HSP90. WO2002/098876 discloses CAK inhibitors. EP778277 discloses CRF antagonists.
While progress has been made in this field, there is a great need to develop a novel compound that inhibits protein kinase. In particular, it would be desirable to develop compounds that inhibit JAK family kinases such as JAK1, JAK2, JAK3 and TYK2. Accordingly, the present invention provides novel compounds which modulate the JAK pathway and are useful for the prevention and/or treatment of auto-immune and/or inflammatory diseases.